The initial differential treatment of the two X chromosomes during X-chromosome inactivation is controlled by the X-inactivation centre (Xic). This locus determines how many X chromosomes are present in a cell ('counting') and which X chromosome will be inactivated in female cells ('choice'). Critical control sequences in the Xic include the non-coding RNAs Xist and Tsix, and long-range chromatin elements. However, little is known about the process that ensures that X inactivation is triggered appropriately when more than one Xic is present in a cell. Using three-dimensional fluorescence in situ hybridization (FISH) analysis, we showed that the two Xics transiently colocalize, just before X inactivation, in differentiating female embryonic stem cells. Using Xic transgenes capable of imprinted but not random X inactivation, and Xic deletions that disrupt random X inactivation, we demonstrated that Xic colocalization is linked to Xic function in random X inactivation. Both long-range sequences and the Tsix element, which generates the antisense transcript to Xist, are required for the transient interaction of Xics. We propose that transient colocalization of Xics may be necessary for a cell to determine Xic number and to ensure the correct initiation of X inactivation.

The effect of trichostatin A (TSA)-induced histone acetylation on the interphase chromatin structure was visualized in vivo with a HeLa cell line stably expressing histone H2A, which was fused to enhanced yellow fluorescent protein. The globally increased histone acetylation caused a reversible decondensation of dense chromatin regions and led to a more homogeneous distribution. These structural changes were quantified by image correlation spectroscopy and by spatially resolved scaling analysis. The image analysis revealed that a chromatin reorganization on a length scale from 200 nm to >1 μm was induced consistent with the opening of condensed chromatin domains containing several Mb of DNA. The observed conformation changes could be assigned to the folding of chromatin during G1 phase by characterizing the effect of TSA on cell cycle progression and developing a protocol that allowed the identification of G1 phase cells on microscope coverslips. An analysis by flow cytometry showed that the addition of TSA led to a significant arrest of cells in S phase and induced apoptosis. The concentration dependence of both processes was studied.

Chromosome capture by microtubules is widely accepted as the universal mechanism of spindle assembly in dividing cells. However, the observed length of spindle microtubules and computer simulations of spindle assembly predict that chromosome capture is efficient in small cells, but may fail in cells with large nuclear volumes such as animal oocytes. Here we investigate chromosome congression during the first meiotic division in starfish oocytes. We show that microtubules are not sufficient for capturing chromosomes. Instead, chromosome congression requires actin polymerization. After nuclear envelope breakdown, we observe the formation of a filamentous actin mesh in the nuclear region, and find that contraction of this network delivers chromosomes to the microtubule spindle. We show that this mechanism is essential for preventing chromosome loss and aneuploidy of the egg--a leading cause of pregnancy loss and birth defects in humans.

Promyelocytic leukemia (PML) and Cajal bodies are mobile subnuclear organelles, which are involved in activities like RNA processing, transcriptional regulation, and antiviral defense. A key parameter in understanding their biological functions is their mobility. The diffusion properties of PML and Cajal bodies were compared with a biochemically inactive body formed by aggregates of murine Mx1 by using single-particle tracking methods. The artificial Mx1-yellow fluorescent protein body showed a very similar mobility compared with PML and Cajal bodies. The data are described quantitatively by a mechanism of nuclear body movement consisting of two components: diffusion of the body within a chromatin corral and its translocation resulting from chromatin diffusion. This finding suggests that the body mobility reflects the dynamics and accessibility of the chromatin environment, which might target bodies to specific nuclear subcompartments where they exert their biological function.C ajal and promyelocytic leukemia (PML) bodies are essential components of the nucleus that are thought to contain activities for RNA processing, transcriptional regulation, and antiviral defense. For understanding their biological functions, parameters have to be identified that characterize their mobility and lead to a localization of Cajal and PML bodies at their target sites in the nucleus. Whereas Cajal bodies are often found associated to several different small nuclear RNA and small nucleolar RNA gene loci as well as histone gene loci (1-5), PML bodies are frequently located in the MHC gene region (6). For both nuclear bodies, different types of movements were described and assigned to distinct subgroups of Cajal and PML bodies (7-12). The analysis of a baby hamster kidney cell line revealed that a fraction of PML bodies moved over longer distances and in an energy-dependent manner. These faster moving bodies were not observed in HeLa cells (8). In a detailed study of Cajal bodies in HeLa cells, an anomalous diffusion behavior and an ATP-and transcription-dependent association with chromatin was reported (7).Here, the mobility of PML and Cajal bodies has been compared with the nuclear body-like structures formed by Mx1 protein fused to yellow fluorescent protein (YFP). Mx proteins are IFN-induced GTPases of the dynamin super family involved in defense mechanisms against RNA viruses (13). The murine Mx1 protein, 72 kDa in size, has a natural nuclear localization sequence sequence and displays a nuclear body-like distribution (14,15). In contrast to the Mx1 WT protein, the Mx1-YFP construct studied has no antiviral activity as tested with influenza and Thogoto virus infection and formed crystals (S. Stertz and O. Haller, personal communication). The diffusion properties of this bona fide biologically inert body were used as a reference to identify principles determining the mobility of bodies within the nucleus. Cell Culture and Transfection. HeLa cells (ATCC CCL-2) were cultured on 12-mm glass coverslips for immunofluorescence or 35-...

We present a simple but accurate algorithm to calculate the flow and shear rate profile of shear thinning fluids, as typically used in biofabrication applications, with an arbitrary viscosity-shear rate relationship in a cylindrical nozzle. By interpolating the viscosity with a set of power-law functions, we obtain a mathematically exact piecewise solution to the incompressible Navier-Stokes equation. The algorithm is validated with known solutions for a simplified Carreau-Yasuda fluid, full numerical simulations for a realistic chitosan hydrogel as well as experimental velocity profiles of alginate and chitosan solutions in a microfluidic channel. We implement the algorithm in an easy-to-use Python tool, included as Supplementary Material, to calculate the velocity and shear rate profile during the printing process, depending on the shear thinning behavior of the bioink and printing parameters such as pressure and nozzle size. We confirm that the shear stress varies in an exactly linear fashion, starting from zero at the nozzle center to the maximum shear stress at the wall, independent of the shear thinning properties of the bioink. Finally, we demonstrate how our method can be inverted to obtain rheological bioink parameters in-situ directly before or even during printing from experimentally measured flow rate versus pressure data.

Active gel theory has recently been very successful in describing biologically active materials such as actin filaments or moving bacteria in temporally fixed and simple geometries such as cubes or spheres. Here we develop a computational algorithm to compute the dynamic evolution of an arbitrarily shaped, deformable thin membrane of active material embedded in a 3D flowing liquid. For this, our algorithm combines active gel theory with the classical theory of thin elastic shells. To compute the actual forces resulting from active stresses, we apply a parabolic fitting procedure to the triangulated membrane surface. Active forces are then dynamically coupled via an Immersed-Boundary method to the surrounding fluid whose dynamics can be solved by any standard, e.g. Lattice-Boltzmann, flow solver. We validate our algorithm using the Green's functions of [Berthoumieux et al. New J. Phys. 16, 065005 (2014)] for an active cylindrical membrane subjected (i) to a locally increased active stress and (ii) to a homogeneous active stress. For the latter scenario, we predict in addition a so far unobserved non-axisymmetric instability. We highlight the versatility of our method by analyzing the flow field inside an actively deforming cell embedded in external shear flow. Further applications may be cytoplasmic streaming or active membranes in blood flows. Published as: Phys. Rev. E 99(6), 062418 (2019)

We investigate the margination of microparticles/platelets in blood flow through complex geometries typical for in vivo vessel networks: a vessel confluence and a bifurcation. Using three-dimensional lattice Boltzmann simulations, we confirm that behind the confluence of two vessels, a cell-free layer devoid of red blood cells develops in the channel center. Despite its small size of roughly 1 μm, this central cell-free layer persists for up to 100 μm after the confluence. Most importantly, we show from simulations that this layer also contains a significant amount of microparticles/platelets and validate this result by in vivo microscopy in mouse venules. At bifurcations, however, a similar effect does not appear, and margination is largely unaffected by the geometry. This antimargination toward the vessel center after a confluence may explain earlier in vivo observations, which found that platelet concentrations near the vessel wall are seen to be much higher on the arteriolar side (containing bifurcations) than on the venular side (containing confluences) of the vascular system.

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